Formulations and methods for generating active cytofectin: polynucleotide transfection complexes

ABSTRACT

In the generation of cytofectin:polynucleotide complexes for transfection of cells, formulations, counterions, and reaction conditions for maximizing the transfection include using a cationic amine compound that has the general structure: ##STR1## wherein R 4  and R 5  are a pair of same or different lipoyl moieties selected from a group consisting of an alkyl, alkenyl, alkynyl, alkanoyl, alkenoyl, or alkynoyl groups and for R 1 , R 2 , and R 3  at least two are hydroxylated, ether containing, or acyloxy containing alkyl, alkenyl, or alkynyl groups or at least one amine bonded halogen containing moiety selected from a group consisting of a halogenated alkyl, alkenyl, or alkynyl group or a mixture of at least one halogen containing moiety selected from a group consisting of a halogenated alkyl, alkenyl, or alkynyl group and at least one hydroxylated, ether containing, or acyloxy containing alkyl, alkenyl, or alkynyl group, and X -   is an oxyanion or halide counterion.

This is a continuation-in-part of U.S. Ser. No. 08/534,471; filed Sep.27, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Provided are formulations and methods of producing activecytofectin:polynucleotide transfection complexes from a collection ofcytofectins (cytofectins are defined as chemical species that arecationic transfection amphiphiles) or cationic lipids that bind andtransport polynucleotides through membrane barriers. More specifically,structural features in cytofectins that improve transfection areselected, cytofectin counterions that improve transfection are chosen,and heating/sonication conditions for the formation oftransfectin:polynucleotide complexes are maximized.

2. Description of the Background Art

Cellular transfection strategies for gene therapy and similar goals havebeen designed and performed, but many of these procedures involverecombinant virus vectors and various problems exist with these viralgene transfer systems. Even generally advantageous adenovirus techniquesencounter difficulties since most humans have antibodies to many of theadenovirus serogroups, including those that have been chosen as vectors.Wild type adenoviral superinfection of an adenoviral vector treatedpatient may result in propagating the recombinant vector as a defectiveviral particle, with the ability to infect many unintended individuals(if chosen to have a rare serogroup). The chance of adenoviralcontamination is quite low but not impossible. The safety of using thesegenetic materials in humans remains unclear and thus hazardous.

Unfortunately, the potential of gene transfer-based research to improvehuman health will be restricted unless improved methods are developedfor in vivo delivery of foreign genetic material into cells and tissues.Currently used viral and non-viral transfection reagents have beencompromised by one or more problems pertaining to: 1) associated healthrisks, 2) immunological complications, 3) inefficient in vivotransfection efficiency, and 4) direct cytotoxicity. The development ofsafe and effective polynucleotide-based medicines will require improvedsolutions which address these problems. Therefore, safe, non-viralvector methods for transfection or gene therapy are essential.

Cationic amphiphiles are currently regarded as an alternative to viralvector technology for in vivo polynucleotide delivery. Cationiclipid-based reagents avoid many of the health and immunological concernsassociated with viral vectors. In a practical sense, cationicamphiphile-based delivery agents are relatively simple to use, and offerunparalleled flexibility in the nature of the material that can bedelivered. Typically, cationic lipid complexes are prepared by mixingthe cationic lipid (cytofectin) with the desired DNA (1), RNA (2),antisense oligomer (3), or protein (4) to yield active particles; incontrast to the laborious recombinant DNA and cell culture manipulationswhich are typically required to produce virus-derived delivery agents.

A few such lipid delivery systems for transporting DNA, proteins, andother chemical materials across membrane boundaries have beensynthesized by research groups and business entities. Most of thesynthesis schemes are relatively complex and generate lipid baseddelivery systems having only limited transfection abilities. A needexists in the field of gene therapy for cationic lipid species that havea high biopolymer transport efficiency. It has been known for some timethat a very limited number of certain quaternary ammonium derivatized(cationic) liposomes spontaneously associate with DNA, fuse with cellmembranes, and deliver the DNA into the cytoplasm (as noted above, thesespecies have been termed "cytofectins"). LIPOFECTIN™ represents a firstgeneration of cationic liposome formulation development. LIPOFECTIN™ iscomposed of a 1:1 formulation of the quaternary ammonium containingcompound DOTMA and dioleoylphosphatidylethanolamine sonicated into smallunilamellar vesicles in water. Problems associated with LIPOCFECTIN™include non-metabolizable ether bonds, inhibition of protein kinase Cactivity, and direct cytotoxicity. In response to these problems, anumber of other related compounds have been developed. The monoammoniumcompounds of the subject invention improve upon the capabilities ofexisting cationic liposomes and serve as a very efficient deliverysystem for biologically active chemicals.

Since the original report (Felgner, P. L., Gadek, T. R., Holm, M.,Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M. andDanielsen, M. 1987. Lipofection: a highly efficient, lipid-mediatedDNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84(21):7413-7, which is herein incorporated by reference, as are all citedreferences in this disclosure) that liposomes comprised of equal amountsof the cytofectin DOTMA (N-1-(2,3-dioleyloxy)propyl!-N,N,N-trimethylammonium chloride) and neutrallipid DOPE (dioleoyl phosphotidylethanolamine) spontaneously associatewith DNA to form efficient transfection complexes, the technology hasadvanced incrementally. There have been few cytofectins developed whichhave improved upon the in vivo activity of the prototypic agent DOTMA.This lack of progress may reflect funding priorities which have focusedon the application of cationic lipid technology to biologic problems,rather than research focusing on principles which effectcytofectin-mediated gene delivery. Specifically, studies focused on themechanism(s) involved in cytofectin actions, barriers tocytofectin-mediated in vivo gene delivery, and clarification ofcytofectin structure/activity relationships would facilitate thedevelopment of improved cationic lipid-based delivery reagents. Whileresearch into the mechanism responsible for cationic amphiphile-mediatedgene delivery is ongoing in a number of laboratories (Sternberg, B.,Sorgi, F. L. and Huang, L. 1994. New structures in complex formationbetween DNA and cationic liposomes visualized by freeze-fractureelectron microscopy. FEBS. Lett. 356(2-3): 361-6, Wrobel, I. andCollins, D. 1995. Fusion of cationic liposomes with mammalian cellsoccurs after endocytosis. Biochim. Biophys. Acta 1235(2): 296-304, andZabner, J., Fasbender, A. J., Moninger, T., Poellinger, K. A. and Welsh,M. J. 1995. Cellular and molecular barriers to gene transfer by acationic lipid. J. Biol. Chem. 270(32): 18997-9007), even the most basicaspects of the mechanism of action of cytofectins (the relativecontributions of direct cytoplasmic membrane fusion and endocytosis)remain unresolved.

Currently, several cationic amphiphile preparations are commerciallyavailable, and new analogs have been published. However, these agentsare frequently reported without comparison to existing compounds, andtherefore it is difficult to derive insights into the relationship ofstructural motifs to polynucleotide transfection. This difficulty hasbeen exacerbated by the variability in: 1) transfected cell typesexploited in the initial report, 2) reporter genes used to characterizetransfection, 3) methods for reporting biologic response (typicallyreporter protein expression) and 4) specific expression vector design.In addition, there have been few reports which describe the effects ofalternative formulation methods. Paradoxically, the relative lack ofsuch fundamental information implies that significant improvements incytofectin-mediated gene transfer technology may be achieved by furthersystematic study.

As indicated above, various cationic lipids have been synthesized inprevious references. In the realm of patents, for example, U.S. Pat. No.4,812,449 discloses in situ active compound assembly of biologicallyactive agents at target locations in preference to surroundings whichare desired to be unaffected. Several charged and uncharged aminederivatives are described.

Introduced in U.S. Pat. No. 5,171,678 are lipopolyamines and their usefor transfecting eukaryotic cells. A polynucleotide is mixed with thesubject lipopolyamine and contacted with the cells to be treated.

U.S. Pat. Nos. 5,186,923 and 5,277,897 relate an enhancement of cellularaccumulation of lipophilic cationic organometallic compounds byreduction of the intramembrane potential. Technetium containingcompounds are disclosed.

Lipophilic cationic compounds are presented in U.S. Pat. No. 5,208,036.Asymmetrical amine compounds are synthesized and employed in a methodfor DNA transfection. The amines are quaternized by two hydrogens oralkyl, aryl, aralkyl, quinuclidino, piperidino, pyrrolidino, ormorpholine groups, unlike the present invention.

U.S. Pat. No. 5,264,618 discloses cationic lipids for intracellulardelivery of biologically active molecules. Asymmetric ammoniumcontaining cationic lipids are presented for transporting molecules intomembrane enclosed systems. The amines are quaternized by two hydrogensor alkyl groups, unlike the present invention.

Transfection of nucleic acids into animal cells via a neutral lipid anda cationic lipid is revealed in U.S. Pat. No. 5,279,833. Liposomes withnucleic acid transfection activity are formed from the neutral lipid andthe ammonium salt containing cationic lipid.

U.S. Pat. No. 5,334,761 describes other amine containing cationiclipids. Cationic lipids are utilized to form aggregates for delivery ofmacromolecules and other compounds into cells. The amines arequaternized by two hydrogens or unbranched alkyl groups, unlike thepresent invention.

In the PCT publication of PCT/US94/13362 a heterocyclic diamine isdisclosed. A symmetrical quaternary diamine having lipid tails isrelated for forming liposomes.

The foregoing patents and publication reflect the state of the art ofwhich the applicants are aware and are tendered with the view towarddischarging applicants' acknowledged duty of candor in disclosinginformation which may be pertinent in the examination of thisapplication. It is respectfully submitted, however, that none of thesepatents teach or render obvious, singly or when considered incombination, applicants' claimed invention.

SUMMARY OF THE INVENTION

An object of the present invention is to disclose formulations,counterions, and conditions that yield lipid:polynucleotide complexesformed from a category of amines that greatly facilitate the delivery ofbiologically active compounds through membrane structures.

Another object of the present invention is to present formulations,counterions, and conditions that yield lipid:polynucleotide complexesformed from a group of cationic amine compounds that assist in thetransport of selected macromolecules and other substances into and pastmembrane barriers.

A further object of the present invention is to relate formulations,counterions, and conditions that yield cytofectin:polynucleotidecomplexes formed from a collection of biologically active moleculetransporters having the general structure: ##STR2## wherein m=1-10; R₁,R₂, and R₃ are the same or different and are hydrogen, an alkyl group,an alkenyl group, an alkynyl group, a hydroxylated alkyl, alkenyl, oralkynyl group, an ether containing alkyl, alkenyl, or alkynyl group, ora halogenated alkyl, alkenyl, or alkynyl group; R₄ is an alkyl group, analkenyl group, an alkynyl group, or an alkyl, alkenyl, or alkynylcontaining acyl group; R₅ is an alkyl group, an alkenyl group, analkynyl group, or an alkyl, alkenyl, or alkynyl containing acyl group;and X⁻ is an anion that assist in the transport of selectedmacromolecules and other substances into and past membrane barriers.

Yet another object of the present invention is to describe formulations,counterions, and conditions that yield cytofectin:polynucleotidecomplexes in which the transfection efficiency is influenced by theeffective sizes of the complexes which in turn are a function of theapplication of sonicating energies and surrounding temperatures.

Still yet another object of the present invention is to disclosestructural properties and counterions of cytofectins that influencetransfection efficiency.

Disclosed are novel formulations, counterions, and heating/sonicationconditions for producing transfection active cytofectin:polynucleotidecomplexes from cationic transporter molecules that facilitate thedelivery of polynucleotides into and beyond membrane barriers orboundaries. Generally related is a cytofectin:polynucleotide complexthat comprises a polynucleotide; a quaternized amine having bonded to anattached carbon chain at least a pair of same or different lipoylmoieties selected from a group consisting of an alkyl, alkenyl, alkynyl,alkanoyl, alkenoyl, or alkynoyl groups and at least two amine bondedhydroxylated, ether containing, or acyloxy containing alkyl, alkenyl, oralkynyl groups or at least one amine bonded halogen containing moietyselected from a group consisting of a halogenated alkyl, alkenyl, oralkynyl group or a mixture of at least one halogen containing moietyselected from a group consisting of a halogenated alkyl, alkenyl, oralkynyl group and at least one hydroxylated, ether containing, oracyloxy containing alkyl, alkenyl, or alkynyl group; and a counterionfor the quarternized amine.

More specifically, compounds having the structure: ##STR3## whereinm=1-10; R₁, R₂, and R₃ are the same or different and are hydrogen, analkyl group, an alkenyl group, an alkynyl group, a hydroxylated alkyl,alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, or a halogenated alkyl, alkenyl, or alkynyl group; R₄ isan alkyl group, an alkenyl group, an alkynyl group, or an alkyl,alkenyl, or alkynyl containing acyl group; R₅ is an alkyl group, analkenyl group, an alkynyl group, or an alkyl, alkenyl, or alkynylcontaining acyl group; and X⁻ is a counterion. Usually, m is 1; R₁, R₂,and R₃ are alkyl groups and R₄ and R₅ are alkyl containing acyl groupsand more commonly m=1; R₁ and R₃ are methyl or equivalent groups; R₂ isan ethyl or equivalent group; and R₄ and R₅ are --CO(CH₂)₁₂ CH₃ orequivalent groups.

The subject cytofectin:polynucleotide complexes are produced bysonicating a mixture of the polynucleotide, the quarternized aminecontaining cytofectin, and the counterion for a selected period of timeat a predetermined temperature. Usually, the selected period of time forsonication is about thirty seconds to about two minutes and thepredetermined temperature is above a phase transition temperature oflipoyl moieties within the cytofectin and is about 40° C. to about 70°C., more usually about 50° C. to about 60° C., and preferably about 56°C.

Counterions that enhance transfection are bisulfate,trifluoromethanesulfonate, and the halides, in particular iodide and toa lesser extent bromide.

Preferred reaction conditions for generating transfection activecytofectin:polynucleotide complexes include elevated temperatures andthe use of sonication during the formation of the complexes.

Other objects, advantages, and novel features of the present inventionwill become apparent from the detailed description that follows, whenconsidered in conjunction with the associated drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of cytofectin-mediated DNA transfection usingNIH 3T3 cells.

FIG. 2 presents an in vivo comparison of cytofectin-mediated DNAtransfection in Balb-C mice.

FIG. 3 shows transfection in vivo data for various amine cytofectins.

FIG. 4 shows the identification of cytofectin structural domains.

FIG. 5; A, B, and C; shows the experimental design for polar domainanalysis in which: A) illustrates an increase in cross-sectional area ofthe polar domain; B) shows an increase in available hydrogen bondingmodes; and C) displays an increase in inductive influence on the chargedcenter.

FIG. 6 shows a comparison of cytofectin polar domain structure totransfection activity in NIH 3T3 cells.

FIG. 7 shows a comparison of cytofectin polar domain structure totransfection activity in vivo for intratracheal instillation into micewith two different hydrophobic side chains.

FIG. 8 shows a comparison of cytofectin counterions to transfectionactivity in NIH 3T3 cells.

FIG. 9 shows a comparison of cytofectin counterions to in vivotransfection activity in Balb-C lung.

FIG. 10 shows a comparison of cytofectin hydrophobic structure totransfection activity in NIH 3T3 cells.

FIG. 11 shows comparison of cytofectin hydrophobic structure totransfection activity in human bronchial epithelial cells (16HBE14o-).

FIG. 12 shows the effects of formulation conditions on luciferaseexpression in murine lung.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Cationic amphiphiles (cytofectins) are widely used for the transfectionof cultured cells, and may become useful for the development of geneticmedicines. Although fundamental research focused on clarification ofphysicochemical structure/biologic function correlations has beenlimited, general principles relating to optimization of cytofectinstructure are beginning to emerge. The formulation studies disclosedhere address the tendency of high concentrationcytofectin:polynucleotide complexes to precipitate. From theseobservations, we show that what we believe to be thermodynamicallystable products can be formed by sonication with heating ofcytofectin:polynucleotide complexes, and that this process reduces thekinetically driven aggregation and precipitation which currentlycomplicates many in vivo studies.

Referring now to the following disclosure and to the data presented inFIGS. 1-11, there are described preferred embodiments of formulationsand conditions that yield cytofectin:polynucleotide complexes formedfrom a cationic monoamine having at least a pair of lipoyl moietiesselected from a group consisting of an alkyl chain, an alkenyl chain,and an alkyl or alkenyl containing acyl chain such as: ##STR4## whereinm=1-10; R₁, R₂, and R₃ are the same or different and are hydrogen, analkyl group, an alkenyl group, an alkynyl group, a hydroxylated alkyl,alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, a halogenated alkyl, alkenyl, or alkynyl group, or acylor acyloxy containing alkyl, alkenyl, or alkynyl group; R₄ is an alkylgroup, an alkenyl group, an alkynyl group, or an alkyl, alkenyl, oralkynyl containing acyl group; R₅ is an alkyl group, an alkenyl group,an alkynyl group, or an alkyl, alkenyl, or alkynyl containing acylgroup; and X⁻ is an anion. The extra, with m more than 1, number ofmethylenes is introduced by standard procedures that complement thedescribed subject synthetic pathways.

A first preferred structure is: ##STR5## wherein for Compound A: n=1-10,usually between 1 and 3, preferably 1; R₃ is a hydrogen, an alkyl group,an alkenyl group, an alkynyl group, or a hydroxylated alkyl, alkenyl,alkynyl group, often an alkyl group of from 1 to 10 carbons, preferablya methyl group; R₄ and R₅ are the same or different with each an alkylgroup, an alkenyl group, an alkynyl group, or an alkyl, alkenyl, oralkynyl containing acyl group; and X⁻ is an anion, usually an oxyanionor halide counterion. ##STR6## where: the abbreviation Tr in thesynthesis scheme denotes --C(Ph)₃, n=1-10, usually between 1 and 3,preferably 1; R₃ is a hydrogen, an alkyl group, an alkenyl group, analkynyl group, or a hydroxylated alkyl, alkenyl, alkynyl group, often analkyl group of from 1 to 10 carbons, preferably a methyl group; R₄ andR₅ are the same or different with each an alkyl group, an alkenyl group,an alkynyl group, or an alkyl, alkenyl, or alkynyl containing acylgroup; and X⁻ is an anion, usually an oxyanion or halide counterion. Itis stressed that although other procedures are contemplated to be withinthe realm of this disclosure, a preferred method for introducingdifferent acyl containing R₄ and R₅ groups in this compound, and in thecompounds below, is the synthesis method given in "A Flexible Approachto Synthetic Lipid Ammonium Salts for Polynucleotide Transfection" byBennett et al. (Tetrahedron Letters, Vol. 36, No. 13, pp. 2207-2210) andis herein incorporated by reference. In this method an acyl migration isemployed to produce the mixed ester products.

In the general synthesis scheme for Compound A derivatives, the firststep involves reacting a tert-butyldiphenylsilyloxy derivatized material(made via a reaction of the dihydroxyethyl starting material withCISiPh₂ tBu) with (triphenylmethoxy)methyloxirane (synthesized accordingto the procedure described in Bennett, M. J., Malone, R. W., and Nantz,M. H. Tetrahedron Lett. 1995, 36, 2207) in the presence of lithiumperchlorate in absolute ethanol. Diethyl ether in formic acid comprisedthe second step. The third step is a reaction with an alkyl, alkenyl, oralkynyl halide or an alkyl, alkenyl, or alkynyl containing acyl halide.The fourth step is tetrabutylammonium fluoride and THF initiated removalof the tert-butyldiphenylsilyloxy protection groups to produce thegeneral precursor compound. The general precursor compound is thenallowed to react with a selected alkyl, alkenyl, alkynyl or hydroxylatedalkyl, alkenyl, or alkynyl halide.

A second preferred structure is: ##STR7## wherein for Compound B:n=1-10, usually between 1 and 3, preferably 1; R₃ is a hydrogen, analkyl group, an alkenyl group, an alkynyl group, or a hydroxylatedalkyl, alkenyl, alkynyl group, often an alkyl group of from 1 to 10carbons, preferably a methyl group; R₄ and R₅ are the same or differentwith each an alkyl group, an alkenyl group, an alkynyl group, or analkyl, alkenyl, or alkynyl containing acyl group; R₆ is an alkyl group,an alkenyl group, an alkynyl group, or an acyl containing group all from1 to 10 carbons, preferably a methyl group; R₇ is an alkyl group, analkenyl group, an alkynyl group, or an acyl containing group all from 1to 10 carbons, preferably a methyl group; and X⁻ is an anion, usually anoxyanion or halide counterion. ##STR8## where: n=1-10, usually between 1and 3, preferably 1; R₃ is a hydrogen, an alkyl group, an alkenyl group,an alkynyl group, or a hydroxylated alkyl, alkenyl, alkynyl group, oftenan alkyl group of from 1 to 10 carbons, preferably a methyl group; R₄and R₅ are the same or different with each an alkyl group, an alkenylgroup, an alkynyl group, or an alkyl, alkenyl, or alkynyl containingacyl group; R₆ is an alkyl group, an alkenyl group, an alkynyl group offrom 1 to 10 carbons, preferably a methyl group; R₇ is an alkyl group,an alkenyl group, an alkynyl group of from 1 to 10 carbons, preferably amethyl group; and X⁻ is an anion, usually an oxyanion or halidecounterion.

In the general synthesis scheme for Compound B the first step involvesreacting an amine starting material with (triphenylmethoxy)methyloxiranein the presence of lithium perchlorate in absolute ethanol. Diethylether in formic acid comprised the second step. The third step is areaction with an alkyl alkenyl, or alkynyl halide or an alkyl, alkenyl,or alkynyl containing acyl halide. The general precursor compound isthen allowed to react with a selected alkyl, alkenyl, alkynyl orhydroxylated alkyl, alkenyl, or alkynyl halide.

A third preferred structure is: ##STR9## wherein for Compound C: a, b,or d are the same or different and are from 0-10, usually between 0 and3, preferably 0 or 1; R₄ and R₅ are the same or different with each analkyl group, an alkenyl group, an alkynyl group, or an alkyl, alkenyl,or alkynyl containing acyl group; R₈, R₉, or R₁₀ are the same ordifferent with each an alkyl, alkenyl, or alkynyl group or halogenatedalkyl, alkenyl, or alkynyl group as long as one is halogen containing;and X⁻ is an anion, usually an oxyanion or halide counterion.

More specifically for Compound C a preferred structure is: ##STR10##wherein for Compound C: a=0-10, usually between 0 and 3, preferably 1;R₄ and R₅ are the same or different with each an alkyl group, an alkenylgroup, an alkynyl group, or an alkyl, alkenyl, or alkynyl containingacyl group; R₈ is a halogenated alkyl, alkenyl, or alkynyl group,preferably a trifluoromethyl group; R₁₁ and R₁₂ are the same ordifferent with each an alkyl, alkenyl, or alkynyl group or halogenatedalkyl, alkenyl, or alkynyl group; and X⁻ is an anion, usually anoxyanion or halide counterion. ##STR11## where: a=0-10, usually between0 and 3, preferably 1; R₄ and R₅ are the same or different with each analkyl group, an alkenyl group, an alkynyl group, or an alkyl, alkenyl,or alkynyl containing acyl group; R₈ is an alkyl, alkenyl, or alkynylgroup or halogenated alkyl, alkenyl, or alkynyl group, preferably atrifluoromethyl group; R₁₁ and R₁₂ are the same or different with eachan alkyl, alkenyl, or alkynyl group or halogenated alkyl, alkenyl, oralkynyl group; and X⁻ is an anion, usually an oxyanion or halidecounterion.

In the general synthesis scheme for Compound C-1 the first step involvesreacting the preferably halogenated starting material with(triphenylmethoxy)methyloxirane in the presence of lithium perchloratein absolute ethanol. A reaction with diethylether in formic acidcomprised the second step. The third step is a reaction with an alkylalkenyl, or alkynyl halide or an alkyl, alkenyl, or alkynyl containingacyl halide. The general precursor compound is then allowed to reactwith a selected alkyl, alkenyl, alkynyl or hydroxylated alkyl, alkenyl,alkynyl, halogenated R₁₁ and R₁₂ that are the same or different witheach an alkyl, alkenyl, or alkynyl group or halogenated alkyl, alkenyl,or alkynyl group halide.

With even more specificity, three preferred structures will now bepresented with specific synthesis schemes (detailed in the Examplesection below).

A first specific preferred structure is: ##STR12##

A second specific preferred structure is: ##STR13##

A third specific preferred structure is: ##STR14##

There are alternate synthesis pathways for the fluorinated derivatives,two of which are presented below, but other pathways, as with the abovesynthesis schemes, are considered within the realm of this disclosure.##STR15##

Compound 17, immediately above, may purchased directly from AldrichChemical Company and is usually ordered from this source. ##STR16##

Note that Compound 19 was prepared from 2,2,2-trifluoroethylamine(Aldrich Chemical Company) according to a literature procedure byWawzonek, S., McKillip, W., and Peterson, C. J. Organic Synthesis, Coll.Vol. V 1973, 758.

General Implications for Synthetic Flexibility

The subject synthesis schemes present opportunities for a widelyflexible array of approaches to synthesizing related amine cationictransport molecules. Not only are monosubstituted amine transporterseasily synthesized by the subject procedures, but so a disubstituted andtrisubstituted derivatives with like or mixed polar domain functionalgroups readily produced. Either a monosubstituted or disubstituted aminestarting material is utilized to generate one or two functional groupsin the final compound or during the quaternization step a functionalgroup containing residue is added (see the fluoronated example above).

By way of example and not by way of limitation, a mixed product issynthesized as follows: ##STR17## wherein R₃₀, R₄₀, and R₅₀ are the sameor different and are a hydrogen, alkyl, alkenyl, or alkynyl group, ahydroxy or ether containing alkyl, alkenyl, or alkynyl group, or ahalogen containing alkyl, alkenyl, or alkynyl group, R₆₀ and R₇₀ arecarbonyl containing or not containing alkyl, alkenyl, or alkynyl groups,and X⁻ is an oxyanion or halide counterion (note that the initialstarting material functional group or groups may need to be protectedvia silation or other appropriate means). More specifically, a preferredsynthesis scheme for a mixed functional product is: ##STR18## whereinR₆₀ and R₇₀ are carbonyl containing or not containing alkyl, alkenyl, oralkynyl groups and X⁻ is an oxyanion or halide counterion.

An example of a synthesis that produces a trisubstituted derivative isas follows: ##STR19##

Cytofectin Structural Domains

Cytofectins can be defined by three principal structural motifs (FIG.4): a cationic polynucleotide binding domain (I), a negatively chargedcounterion (II), and a hydrophobic domain (III). The chemical nature ofthese domains dictates the biophysical properties exhibited by thecytofectins. Thus, structural modifications within each motif can resultin significant alterations in the behavior of pharmaceuticals containingthese amphiphiles. For this reason, researchers have attempted tocorrelate biophysical properties, compound structure, and functionalassessments of polynucleotide transfection.

Polar Domain Structural Considerations

In general, various functionalities have been incorporated intocytofectin polar domains. Many of these functionalities are known tomodulate the binding and condensation of polynucleotides intocytofectin:polynucleotide complexes. These polynucleotide bindingdomains, usually comprised of nitrogen-based groups, are cationic eitheras a consequence of their basicity in aqueous solutions, or viaN-alkylation to yield quaternary amines. Researchers have preparedseveral cytofectins which contain a variety of nitrogen-basedfunctionality including: tetraalkylammonium (Felgner, P. L., Gadek, T.R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P.,Ringold, G. M. and Danielsen, M. 1987. Lipofection: a highly efficient,lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A.84(21): 7413-7, Leventis, R. and Silvius, J. R. 1990. Interactions ofmammalian cells with lipid dispersions containing novel metabolizablecationic amphiphiles. Biochim. Biophys. Acta 1023(1): 124-32, andFelgner, J. N., Kummar, R., Sridhar, C. N., Wheeler, C., Tsai, Y. J.,Border, R., Ramsay, P., Martin, M. and Felgner, P. 1994. Enhanced genedelivery and mechanism studies with a novel series of cationic lipidformulations), polyammonium (Behr, J. P., Demeneix, B., Loeffler, J. P.and Perez-Mutul, J. 1989. Efficient gene transfer into mammalian primaryendocrine cells with lipopolyamine-coated DNA. Proc. Nati. Acad. Sci.U.S.A. 86(18): 6982-6, Zhou, X. H., Klibanov, A. L. and Huang, L. 1991.Lipophilic polylysines mediate efficient DNA transfection in mammaliancells. Biochim. Biophys. Acta 1065(1): 8-14, and Puyal, C., Milhaud, P.,Bienvenue, A. and Philippot, J. R. 1995. A new cationic liposomeencapsulating genetic material. A potential delivery system forpolynucleotides. Eur. J. Biochem. 228(3): 697-703), monoalkylammonium(Gao, X. A. and Huang, L. 1991. A novel cationic liposome reagent forefficient transfection of mammalian cells. Biochem. Biophys. Res.Commun. 179(1): 280-5), and amidine-based (Ruysschaert, J. M., elOuahabi, A., Willeaume, V., Huez, G., Fuks, R., Vandenbranden, M. and DiStefano, P. 1994. A novel cationic amphiphile for transfection ofmammalian cells. Biochem. Biophys. Res. Commun. 203(3): 1622-8). Suchfunctionality may have an influence on the efficiency with whichpolynucleotides interact with cationic lipid particles, the interactionsbetween lipid/DNA complexes and biological membranes, and themechanism(s) by which these complexes deliver polynucleotides into thecells. Therefore, studies correlating polar domain chemical structureand physical properties with transfection activity would be predicted toclarify the role of polar domain hydration and intermolecular bonding onpolynucleotide delivery. Such a study is presented herein.

A number of investigations into polar domain structure/activityrelationships have been reported. Previous studies have focused on theoptimization of the alkyl chain length separating the lipidic domain andpolynucleotide binding group (Ito, A., Miyazoe, R., Mitoma, J., Akao,T., Osaki, T. and Kunitake, T. 1990. Synthetic cationic amphiphiles forliposome-mediated DNA transfection. Biochem. Int. 22(2): 235-41 andFarhood, H., Bottega, R., Epand, R. M. and Huang, L. 1992. Effect ofcationic cholesterol derivatives on gene transfer and protein kinase Cactivity. Biochim. Biophys. Acta 1111(2): 239-46); correlations betweencationic functionality (tetraalkylammonium vs. trialkylammonium),protein kinase C activity, and transfection efficacy (Farhood, H.,Bottega, R., Epand, R. M. and Huang, L. 1992. Effect of cationiccholesterol derivatives on gene transfer and protein kinase C activity.Biochim. Biophys. Acta 1111(2): 239-46); and correlations betweenheadgroup charge density and transfection activity (Remy, J. S., Sirlin,C., Vierling, P. and Behr, J. P. 1994. Gene transfer with a series oflipophilic DNA-binding molecules. Bioconjug. Chem. 5(6): 647-54).Subsequently, the subject research was conducted to explore thecorrelations between transfection activity and functional modificationsof the tetraalkylammonium moiety used to bind anionic regions of DNA,RNA, and related polymers. The decision to usetetraalkylammonium-derivatized cytofectins for this study was based onunpublished observations that tertiary ammonium analogs of DOTMA-relatedcompounds are not active transfection agents.

Evidence supporting the hypothesis that modifications in cytofectinheadgroup structure (and associated physical properties) can influencetransfection activity comes from research performed by Feigner et al.(Felgner, J. N., Kummar, R., Sridhar, C. N., Wheeler, C., Tsai, Y. J.,Border, R., Ramsay, P., Martin, M. and Felgner, P. 1994. Enhanced genedelivery and mechanism studies with a novel series of cationic lipidformulations. J. Biol. Chem. 269(4): 2550-2561) and our laboratories(Bennett, M. J., Malone, R. W. and Nantz, M. H. 1995. A flexibleapproach to synthetic lipid ammonium salts for polynucleotidetransfection. Tetrahedron Lett. 36(1): 2207-2210 and Balasubramaniam, R.P., Bennett, M. J., Aberle, A. M., Malone, J. G., Nantz, M. H. andMalone, R. W. 1996. Structural and functional analysis of cationictransfection lipids: the hydrophobic domain. Gene Ther. 3(2): 163-172).These studies have shown that cytofectins which incorporate ahydroxyethyl-derivatized tetraalkylammonium group in their polar domaindemonstrate enhanced transfection activity when compared to analogswhich do not incorporate such groups (e.g., DORI vs. DOTAP).

We postulate that variations in the chemical composition of thetetraalkylammonium group of cytofectins might effect transfectionactivity by: 1) influencing cationic liposome/polynucleotideinteractions during formulation, 2) altering interactions betweencationic liposome/polynucleotide complexes and cell membranes, 3)altering pathways by which these complexes enter cells, 4) alteringintracellular trafficking of lipid:polynucleotide complexes, and 5)altering the disassociation of the lipid:polynucleotide complex. Wehypothesize that the covalent attachment of select functional groups,like the hydroxyethyl group, to the ammonium moiety can influence bothlipid surface hydration and the effective charge of the ammonium group.These effects can alter the cytofectins' transfection activity byinfluencing one or more of the above processes.

Surface hydration may be one important property by which modifying thepolar domain may alter cytofectin transfection activity. The degree oflipid surface hydration can influence intermolecular lipid interactionsboth prior to and following addition of polynucleotide. Such effectsmight occur via a variety of interactions. The addition of alkyl groupsof increasing chain length to the ammonium group could weakenintermolecular lipid interactions by increasing the cross-sectional areaof the headgroup (steric effects). The presence or absence of functionalgroups which can participate in hydrogen bond formation as eitheracceptors or donors will effect hydration, interaction withpolynucleotide, and bonding to adjacent lipids (e.g. cytofectins, DOPEor cellular lipids). The inclusion of electron withdrawing functionalitymay influence the effective cationic charge of the binding domainthrough an inductive effect. However, these complex interactions make itdifficult to identify the principal factors influencing cytofectintransfection activity. An example is the inclusion of functional groupscapable of hydrogen bonding. In this example, either stronger or weakerlipid-lipid interactions might arise as a consequence of 1)intermolecular hydrogen bonding or 2) increased headgroup hydrationrespectively. Thus, we chose to empirically analyze the effect of suchalterations on the DNA transfection activity.

In order to test hypotheses pertaining to lipid surface hydration andeffective cationic charge, a panel of cationic amphiphiles (see FIG. 5)were prepared by simple N-alkylation ofN,N-dimethyl-1,2-dimyristoyloxy-3-aminopropane with the correspondingalkyl halide, using a procedure analogous to that used by Felgner et al.(Felgner, J. N., Kummar, R., Sridhar, C. N., Wheeler, C., Tsai, Y. J.,Border, R., Ramsay, P., Martin, M. and Felgner, P. 1994. Enhanced genedelivery and mechanism studies with a novel series of cationic lipidformulations. J. Biol. Chem. 269(4): 2550-2561). Lipid thin-filmscontaining these amphiphiles and an equal molar amount of DOPE werehydrated using sterile deionized water, mixed with the plasmid DNApNDCLUX (Aberle, A. M., Bennett, M. J., Malone, R. W. and Nantz, M. H.1996. The counterion influence of cationic lipid-mediated transfectionof plasmid DNA. Biochim. Biophys. Acta 1299(3): 281-283), (encoding theP. pyralis luciferase) and the resulting complexes were used totransfect NIH 3T3 murine fibroblast cells. The experimental design isoutlined in FIG. 5. Based on the transfection data obtained from thisexperiment (see FIG. 6), the following conclusions can be made: 1) Ofthe lipids selected to examine correlations between lipid polar domaincross sectional area and transfection activity, DMEAP (see Example 19below and FIG. 5 for the exact structure of this cytofectin and thecytofectins referred to below) had the highest levels of plasmidtransfection activity. 2) Of the lipids selected to test the influencesof hydrogen bonding, both the methyl ether containing lipid DMMEP andthe hydroxyethyl containing lipid showed a 2-fold enhancement overDMPAP, which has no hydrogen bonding capabilities. We observed nocorrelation between the number or modes of hydrogen bonding (acceptorvs. donor). Polar domain influences on the transfection ability ofselected compounds are presented below (see FIGS. 6, in vitro, and 7, invivo).

Counterion Considerations

All positively charged cytofectin polar domains incorporatingmonoalkylammonium, polyammonium, or tetraalkylammonium-basedfunctionality, have an associated negatively charged counterion.Monoalkylammonium and polyammonium-containing lipids typically have oneor more hydroxide counterions as a consequence of ammonium saltformation resulting from the basicity of the corresponding primary,secondary, or tertiary amine in aqueous media. Tetraalkylammonium-basedcytofectins acquire their negatively charged counterion when theammonium salt is formed as a result of N-alkylation.

As a result of the association of the negatively charged counterion withthe positively charged polynucleotide binding domain, we believe thatthe chemical nature of the counterion may effect the physicochemicalproperties of cytofectins. Specifically, the counterion could influencelipid surface hydration, vesicle fluidity, and lipid polymorphism.Therefore, the counterion could also influence the transfection activityof cytofectins.

In order to study possible counterion influences on cytofectinmediatedtransfection activity, a panel of DOTAP (N-1,2,3-dioleoyloxy)propyl!-N,N,N-trimethylammonium) analogs, differingonly in the anionic counterion, were prepared using ion exchangechromatography (Aberle, A. M., Bennett, M. J., Malone, R. W. and Nantz,M. H. 1996. The counterion influence of cationic lipid-mediatedtransfection of plasmid DNA. Biochim. Biophys. Acta 1299(3): 281-283).The series of counterions (Table 1) were selected so that directcomparisons between anion-water interactions could be made. Previousstudies have shown that there is a correlation between membrane behaviorin various salt solutions and the nature of the anions categorizedaccording to the Hofmeister series (Epand, R. M. and Bryszewska, M.1988. Modulation of the bilayer to hexagonal phase transition andsolvation of phosphatidylethanolamines in aqueous salt solutions.Biochemistry 27(24): 8776-9, Koynova, R. D., Tenchov, B. G. and Quinn,P. J. 1989. Sugars favour formation of hexagonal (HII) phase at theexpense of lamellar liquid-crystalline phase in hydratedphosphatidylethanolamines. Biochim. Biophys. Acta 980: 377-380, andCollins, K. D. and Washabaugh, M. W. 1985. The Hofmeister effect and thebehaviour of water at interfaces. Q. Rev. Biophys. 18(4): 323-422),which groups ions as either kosmotropes (water structuring) orchaotropes (water destabilizing) (see Table 1).

                  TABLE 1    ______________________________________    Summary of cytofectin counterions    Kosmotropes          Chaotropes    ______________________________________    HSO.sub.4.sup.-1     I.sup.-    CF.sub.3 SO.sub.3.sup.-1                         Br    H.sub.2 PO.sub.4     Cl.sup.-    SO.sub.4.sup.-2      CH.sub.3 C(O)O.sup.-    ______________________________________

Transfection analyses using these cytofectins were performed in vitro(NIH 3T3 murine fibroblasts), and in vivo (intratracheal instillationinto mice) with excellent correlation between the in vitro (FIG. 8) andin vivo (FIG. 9) data. One may infer that the trends observed in thesescreenings may be applicable to a variety of cell types. Furthermore,the data suggests that the highly delocalized polar kosmotropicoxyanions, bisulfate and trifluoromethanesulfonate (triflate), promotethe highest levels of luciferase expression. Among the halogensexamined, the DOTAP iodide analog was the most active. It is believedthat iodide most closely associates with the alkylammonium headgroup dueto electrostatic interactions, while oxyanions competitively bind wateraway from the lipid surface. Thermodynamic arguments have suggested thatlipid:solvent interactions directly influence lipid polymorphism. Thus,there may be an exclusion of water and closer interchain packing. Inconclusion, these results indicate that incorporation of anions whichcan facilitate dehydration of the cytofectin polar domain leads toincreased cytofectin transfection activity.

Hydrophobic Domain Structural Considerations

There are two types of hydrophobic domains which have been used in thedesign of cytofectins; sterol-based and di-acyl/alkyl-based domains. Thehydrophobic domain, which can serve as a scaffold from which a lipidbilayer structure is built, also modulates bilayer fluidity and lipidpolymorphism. Increased bilayer fluidity can lead to more efficientformation of lipid:DNA complexes and enhanced fusion of cytofectin:DNAcomplexes with cell or endosomal membranes, which is likely to be a keymechanistic step of the transfection process. While the contribution ofsterol hydrophobic domains to bilayer fluidity is primarily dependent onthe relative concentration of the sterol in the lipid particle, it isthe chemical structure of the aliphatic groups contained indi-acyl/alkyl-based lipids which dictate their contribution to membranefluidity.

It has been previously stated that there is a direct correlation betweencytofectin transfection activity and transfection lipid bilayerfluidity. This hypothesis was originally forwarded by Akao et al. (Akao,T., Osaki, T., Mitoma, J., Ito, A. and Kunitake, T. 1991. Correlationbetween Physicochemical Characteristics of Synthetic CationicAmphiphiles and Their DNA Transfection Ability. Bull. Chem. Soc. Jpn.64: 3677), and suggests that one primary requirement of an amphiphilefor DNA transfection is that the T_(c) (phase transition temperaturebetween the gel and liquid crystalline phases) be lower than 37° C., sothat the transfection lipid assumes a fluid liquid crystalline state atcell culture temperatures. Research supporting this hypothesis (Felgner,J. N., Kummar, R., Sridhar, C. N., Wheeler, C., Tsai, Y. J., Border, R.,Ramsay, P., Martin, M. and Felgner, P. 1994. Enhanced gene delivery andmechanism studies with a novel series of cationic lipid formulations. J.Biol. Chem. 269(4): 2550-2561 and Akao, T., Osaki, T., Mitoma, J., Ito,A. and Kunitake, T. 1991. Correlation between PhysicochemicalCharacteristics of Synthetic Cationic Amphiphiles and Their DNATransfection Ability. Bull. Chem. Soc. Jpn. 64: 3677) has relied onanalysis of a limited number of cytofectin analogs and cell lines. Ininterpreting such studies, we believe it is important that results beobtained using multiple cell lines or tissues before drawing generalconclusions correlating hydrophobic domain structure, bilayer physicalproperties, and transfection activity (Balasubramaniam, R. P., Bennett,M. J., Aberle, A. M., Malone, J. G., Nantz, M. H. and Malone, R. W.1996. Structural and functional analysis of cationic transfectionlipids: the hydrophobic domain. Gene Ther. 3(2): 163-172). It should benoted that, as of now, there is no evidence that the fluidity of neatcytofectin lipids predicts the fluidity of lipidic structures when boundto polynucleotide.

The bilayer fluidity of liposomes containing di-acyl/alkyl-basedcytofectins can be modified by manipulating the symmetry (see Table 2),chain length, and saturation of the aliphatic groups contained in theselipids. In order to understand the relationships between cytofectinhydrophobic domain structure and transfection activity, we prepared apanel of cytofectins differing only in the composition of the aliphaticgroups contained in the hydrophobic domain (Balasubramaniam, R. P.,Bennett, M. J., Aberle, A. M., Malone, J. G., Nantz, M. H. and Malone,R. W. 1996. Structural and functional analysis of cationic transfectionlipids: the hydrophobic domain. Gene Ther. 3(2): 163-172). Cytofectinsexamined in this study included compounds with both symmetric anddissymmetric hydrocarbon side chains which varied in length from C18:1to C8:0. A previous report (Felgner, J. N., Kummar, R., Sridhar, C. N.,Wheeler, C., Tsai, Y. J., Border, R., Ramsay, P., Martin, M. andFeigner, P. 1994. Enhanced gene delivery and mechanism studies with anovel series of cationic lipid formulations. J. Biol. Chem. 269(4):2550-2561) also examined a similar series of cytofectins. However, onlysymmetric hydrocarbon side chains varying in length form C18:1 to C14:0were examined using a single cell line (COS-7). This previous studyindicated that the dimyristyl-containing compound DMRIE was mosteffective for DNA transfection of COS-7 cells. Since shorter side chainswere not examined, the minimal effective acyl chain length was notdefined. Cationic liposomes containing these cytofectins were formulatedusing 1:1 molar ratios of the cytofectin and DOPE. Transfection studiesusing NIH 3T3 murine fibroblast cells (see FIG. 10), CHO cells, and acultured respiratory epithelial cell line (16HBE14o-) (see FIG. 11)revealed some intriguing observations. These are: 1) no single symmetricor dissymetric analog was most effective for DNA transfection of eithercell line examined, 2) dissymmetric lipids resulted in levels ofluciferase expression that were equal to or better than the most activesymmetric lipid analogs, 3) dissymmetric cytofectins with shorter sidechains (C12:0, C14:0) were among the most active lipids in the celllines screened, and 4) the dioctanoyl (di C8:0) compound was generallythe least active, indicating that the effective lower limit of fattyacyl chain length is defined at C(12).

                  TABLE 2    ______________________________________    Summary of symmetric and dissymmetric cytofectins.    1 #STR20##    Cytofectin  R.sup.1       R.sup.2    ______________________________________    DORI        Oleoyl (18:1) Oleoyl (18:1)    DPRI        Palmitoyl (16:0)                              Palmitoyl (16:0)    DMRI        Myristoyl (14:0)                              Myristoyl (14:0)    DLRI        Lauroyl (12:0)                              Lauroyl (12:0)    DO'RI       Octanoyl (8:0)                              Octanoyl (8:0)    OPRI        Oleoyl (18:1) Palmitoyl (16:0)    PORI        Palmitoyl (16:0)                              Oleoyl (18:1)    OO'RI       Oleoyl (18:1) Octanoyl (8:0)    O'ORI       Octanoyl (8:0)                              Oleoyl (18:1)    MLRI        Myristoyl (14:0)                              Lauroyl (12:0)    LMRI        Lauroyl (12:0)                              Myristoyl (14:0)    ______________________________________

Formulation Considerations

Cytofectins spontaneously form transfection complexes with a variety ofbiological polymers upon mixing in aqueous solvent. The mixing orformulation protocols used to prepare active transfection complexescurrently involve optimization of; 1) the ratio of cationiclipid:neutral lipid, 2) solvent type, and 3) the molar ratio of cationiccharge:polynucleotide phosphate charge.

Many optimized formulations incorporateDioleoylphosphatidyl-ethanol-amine (DOPE) along with the cytofectinprior to mixing with DNA, although the high activity frequently observedwith neat DOTAP indicates that DOPE is not always required. DOPE isknown to be a strong destabilizer of lipid bilayers (Litzinger, D andHuang, L. 1992. Phosphatidylethanolamine liposomes: drug delivery, genetransfer and immunodiagnostic applications. Biochim. Biophys. Acta 1113,201-227), and hence can enhance the intrinsic fusogenic properties ofmany cytofectins. Empirical optimization of cytofectin:DOPE molar ratiofor various cell lines, tissues, and cytofectins can result in markedenhancement of transfection activity for the chosen application. In ourhands, the optimized molar ratio has ranged from 9:1 to 1:2(cytofectin:DOPE).

Cell culture experiments typically employ a formulation solventconsisting of either the media in which the cell line is cultured, orOptiMem (Gibco/BRL), a serum-free media which is enriched in factorsincluding transferrin and various growth factors. The enhancedtransfection activity which can be observed with OptiMem may reflectincorporation of the added biologically active agents into thelipid:polynucleotide complex. In such cases, binding to cell surface maybe facilitated by specific ligand:receptor interactions. Complexes aretypically prepared for in vivo administration using either water forinjection or isotonic solvents such as physiologic saline. In contrastto cell culture results, solvent-specific enhancement has not beenreported.

Molar cytofectin:polynucleotide phosphate charge ratio employed duringformulation is frequently not reported, but typically ranges from 1:1 to4:1 for cultured cells. Protocols for in vivo application, andparticularly for pulmonary transfection, frequently employ strikinglydifferent molar charge ratios. Yoshimura (Yoshimura, K., Rosenfeld, M.A., Nakamura, H., Scherer, E. M., Pavirani, A., Lecocq, J. P. andCrystal, R. G. 1992. Expression of the human cystic fibrosistransmembrane conductance regulator gene in the mouse lung after in vivointratracheal plasmid-mediated gene transfer. Nucleic Acids Res. 20(12):3233-40) first described the use of very low cytofectin:DNA chargeratios for pulmonary delivery, and performed an in vivo charge titrationranging from approximately 1:2.5 to 1:35. Optimal activity was obtainedusing the 1:35 ratio of cytofectin:DNA charge. This in vivo protocolalso employed up to 1.4 milligram of plasmid/200 microliter injection, a1,000 to 10,000 fold higher concentration of polynucleotide than istypically used for transfection of cultured cells. As the directinjection of free plasmid DNA into murine lung can result in significantlevels of reporter gene expression (Yoshimura, K., Rosenfeld, M. A.,Nakamura, H., Scherer, E. M., Pavirani, A., Lecocq, J. P. and Crystal,R. G. 1992. Expression of the human cystic fibrosis transmembraneconductance regulator gene in the mouse lung after in vivo intratrachealplasmid-mediated gene transfer. Nucleic Acids Res. 20(12): 3233-40,Meyer, K. B., Thompson, M. M., Levy, M. Y., Barron, L. G. and Szoka, F.C. 1995. Intratracheal gene delivery to the mouse airway:characterization of plasmid DNA expression and pharmacokinetics GeneTher. 2(7): 450-60, and Balasubramaniam, R. P., Bennett, M. J., Aberle,A. M., Malone, J. G., Nantz, M. H. and Malone, R. W. 1996. Structuraland functional analysis of cationic transfection lipids: the hydrophobicdomain. Gene Ther. 3(2): 163-172), it is conceivable that complexesformulated with very low cytofectin:DNA ratios are not the activeprinciple in the observed pulmonary transfections. We hypothesize thatthe transfection activity observed by Yoshimura et al. reflects theactivity of unbound or minimally bound polynucleotide. In this case, theenhanced transfection activity which was observed upon adding smallquantities of lipofectin to concentrated plasmid DNA may reflect partialprotection from nucleases, rather than lipid-mediated transfection whichoccurs in cell culture. We have observed that similar charge titrationexperiments employing 20 micrograms of plasmid DNA/200 microlitersresult in optimized cytofectin:DNA charge ratios of 2:1 to 3:1,depending on cytofectin type and DOPE ratio.

High concentration lipid:DNA complexes (2:1 lipid:DNA charge ratio, 0.1mg/ml or greater) are relatively insoluble, and tend to precipitateduring formulation (see Table 3). We have observed that suchprecipitates are relatively inactive as transfection agents both in cellculture and in vivo. Furthermore, even apparently stablelipid:polynucleotide emulsions can precipitate when stored at roomtemperature for prolonged periods. Vortex mixing and heating tend tofacilitate the precipitation of high concentration complexes. Theprecipitation of cytofectin:DNA complexes represents a significantobstacle to the development of cytofectin medicines. We hypothesize thatthese precipitates represent a kinetic product of lipid:DNA associationrather than the thermodynamically stable product which forms upon mixingat low concentration. This hypothesis follows from the followingmodel: 1) During initial lipid:DNA binding, DNA induces lipidreorganization (polynucleotide coating), resulting in DNA condensationand/or particle restructuring (complex maturation-formation of thethermodynamic product). There are energetic barriers to suchrestructuring which reflect lipid:lipid interactions, displacement ofthe counterion during polynucleotide binding, lipid:polynucleotidebinding, and alterations in polynucleotide hydration duringcondensation. Therefore, the rate of such reorganization will be afunction of lipid structure, counterion type, polynucleotide structure,and temperature. 2) Partially reorganized complexes are subject toaggregation upon collision in solution. This aggregation (e.g. crosslinked proto-complexes- a kinetic product) may be mediated via bindingof uncoated polynucleotide by uncomplexed cytofectin present on thesurface of the colliding particle. Therefore, we predict thataggregation will be a function of concentration, system energy(temperature, vortexing), solution viscosity, and time.

For Table 3, the dynamic light scattering (DLS) estimates of sonicatedversus unsonicated DNA-lipid complexes were for increasing plasmid DNAconcentrations at a fixed 2:1 lipid-DNA charge ratio. Sizing experimentswere designed to mimic murine lung transfection conditions. The complexwas sonicated briefly (30" to 2 minutes) using a bath sonicator(Laboratory equipment, Hicksville NY or the equivalent) at 56° C. (abovethe phase transition temperature of the lipoyl moieties within thecytofectin) until visible aggregates were dispersed. DLS experimentswere performed using a Brookhaven Instruments BI-90 particle sizer, orthe equivalent, at 25° C., sampled continuously for five minutes andanalyzed by the methods of cumulants. No significant shift in particlesize distribution was observed over time (data not shown).

                  TABLE 3    ______________________________________    Effect of Formulation with Heating and Sonication on    Cytofectin: DNA Particle Size.    Sonicated          Unsonicated     DNA! effective polydispersity                               effective                                        polydispersity    mg/ml diameter  index      diameter index    ______________________________________    0.1   400       0.268      3900     1.548    0.2   300       0.019      2800     1.413    0.4   288       0.092      5700     3.682    0.8   334       0.154      2268     2.094    1.0   929       0.685      2110     1.800    ______________________________________

Methods for overcoming the aggregation and precipitation of highconcentration complexes would greatly facilitate preparation ofcytofectin-based genetic medicines. Unfortunately, adding thermal energywould be predicted to both facilitate "maturation" and to increasediffusion within the system, thereby increasing collision rate andenergy. We hypothesize that heating combined with either an increase insystem viscosity or use of sonication to rapidly resolve aggregatesprior to restructuring and precipitation will favor formation of thethermodynamic product. As demonstrated in Table 3 above, the combinationof heating with sonication does result in the formation of stable,smaller lipidic particles. Furthermore, this process appears to enhancethe pulmonary transfection activity of a range of lipids (see FIG. 12),including the relatively inactive compound DOTAP.

EXAMPLES Example 1 Synthesis of N,N-Bis(2-tert-butyldiphenylsilyloxyethyl)!amine, Compound 2 in an aboveSpecific Synthesis Scheme

To a mixture of diethanolamine (4.78 g, 7.26 mmol), triethylamine (2.5mL), and 4-dimethylaminopyridine (89 mg, 0.73 mmol) in dichloromethane(73 mL) at 0° C. was added tert-butylchlorodiphenylsilane (5.46 g, 18.14mmol). On complete addition, the reaction mixture was allowed to warm toroom temperature. After 12 h, the reaction mixture was transferred to aseparatory funnel and the organic layer was washed successively withsaturated aqueous sodium bicarbonate, water, and brine. The organiclayer was dried (sodium sulfate), filtered, and the filtrate solventremoved in vacuo. The crude product so obtained was purified by silicagel column chromatography (1% methanol in dichloromethane) to yield 2.53g (2.13 mmol, 29%) of 2 as an oil. ¹ H NMR (300 MHz, CDCl₃) d 7.70-7.34(m, 20H), 3.79 (t, J=5 Hz, 4H), 2.79 (t, J=5 Hz, 4H), 2.09, (s, 1H),1.05 (s, 18H); ¹³ C NMR (75 MHz, CDCl₃) d 135.5, 133.6, 129.6, 127.6,63.5, 51.7, 26.9, 19.2; IR (KBr) 3071, 2930, 1428 cm⁻¹.

Example 2 (±)- (Triphenylmethoxy)methyl!oxirane, Compound 3 in an aboveSpecific Synthesis Scheme

To a mixture of (±) glycidol (4.00 g, 33.5 mmol), triethylamine (5.7mL), and 4-dimethylaminopyridine (420 mg, 3.40 mmol) in dichloromethane(170 mL) at 0° C. was added triphenylmethyl chloride (16.5 g, 51.2mmol). On complete addition, the reaction mixture was allowed to warm toroom temperature. After 12 h, the reaction mixture was transferred to aseparatory funnel and the organic layer was washed successively withsaturated aqueous sodium bicarbonate, water, and brine. The organiclayer was dried (sodium sulfate), filtered, and the filtrate solventremoved in vacuo. The crude product so obtained was purified by silicagel column chromatography (3% diethylether in hexane) to yield 8.70 g(27.5 mmol, 82%) of 3 as an oil. ¹ H NMR (300 MHz, CDCl₃) d 7.47-7.20(m, 15H), 3.33-3.30 (m, 1 H), 3.16-3.09 (m, 3H), 2.76 (m, 1H), 2.61 (dd,J=2, 5H, 1H); ¹³ C NMR (75 MHz, CDCl₃) d 143.8, 128.6, 127.9, 127.8,127.1, 127.0, 86.7, 64.7, 51.0, 44.6; IR (KBr) 3057, 2922, 1448 cm⁻¹.

Example 3 (±)-3- N,N-bis(2-tertbutyldiphenylsilyloxyethyl)amino!-1-(Triphenylmethoxy)-2-propanol,Compound 4 in an above Specific Synthesis Scheme

To a mixture of (±)-(triphenylmethoxy)methyloxirane (7.66 g, 24.2 mmol)and lithium perchlorate (5.87 g, 55.2 mmol) in absolute ethanol (110 mL)was added amine 2 (11.7 g, 20.2 mmol). The reaction mixture was warmedto 65° C. and allowed to stir for 24 h. After this time, the reactionsolution was allowed to cool to room temperature and then transferred toa separatory funnel containing diethylether (100 mL). The resultantmixture was sequentially washed with saturated aqueous sodiumbicarbonate, water, and brine. The organic layer was dried over sodiumsulfate, filtered and the filtrate was concentrated by rotaryevaporation to give the crude product as a yellow oil. Purification wasaccomplished by SiO₂ column chromatography (3% methanol indichloromethane) to yield 14.5 g (16.1 mmol, 80%) of 4 as an oil. ¹ HNMR (300 MHz, CDCl₃) d 7.65-7.20 (m, 25H), 3.73-3.56 (m, 5H), 3.17 (dd,J=5, 9 Hz, 1H), 2.97 (dd, J=5, 9 Hz, 1H), 2.69 (m, 5H), 2.45 (dd, J=10,12 Hz, 1H), 1.02 (s, 18H); ¹³ C NMR (75 MHz, CDCl₃) d 144.1,135.5,134.7, 133.5, 129.6, 128.7, 128.6, 127.7, 127.6, 126.8, 86.4,67.2, 66.1, 62.1, 58.4, 56.6, 26.8, 26.5, 19.0; IR (KBr) 3445, 3069,2930, 1427 cm⁻¹.

Example 4 (±)-3-N,N-Bis(2-tert-butyldiphenylsilyloxyethyl)amino!-1,2-propanediol,Compound 5 in an above Specific Synthesis Scheme

To a mixture of amine 4 (8.43 g, 9.40 mmol) in diethylether (12 mL) wasadded 85% formic acid (35 mL). The resulting reaction mixture wasstirred at room temperature for 20 h. After this time, solid NaHCO₃ wasadded to neutralize the acidic solution. The resultant mixture wassubsequently diluted with diethylether (100 mL) and transferred to aseparatory funnel. The organic layer was separated and sequentiallywashed with water, and brine. Purification was accomplished by SiO₂column chromatography (3% methanol in dichloromethane) to yield 3.75 g(5.73 mmol, 61%) of 5 as an oil. ¹ H NMR (300 MHz, CDCl₃) d 7.65-7.31(m, 20H), 3.68-3.60 (m, 6H), 3.40 (dd, J=4, 9 Hz, 1H), 2.71 (m, 4H),2.57 (d, J=7 Hz, 2H), 1.03 (s, 18H); ¹³ C NMR (75 MHz, CDCl₃) d 135.5,133.4, 129.7, 127.7, 68.0, 64.4, 62.0, 57.2, 56.7, 26.8, 19.0; IR (KBr)3432, 3070, 2931, 1428 cm⁻¹.

Example 5 (±)-3- N, N-Bis(2-tert-butyidiphenylsilyloxyethyl)amino!1,2-bis(9(z)-octadecenoyloxy)propane,Compound 6 in an above Specific Synthesis Scheme

To a mixture of diol 5 (4.78 g, 7.26 mmol), triethylamine (2.5 mL), and4-dimethylaminopyridine (89 mg, 0.73 mmol) in dichloromethane (73 mL) at0° C. was added dropwise oleoyl chloride (5.46 g, 18.14 mmol). Oncomplete addition, the reaction mixture was allowed to stir at 0° C. for4 h whereupon an additional portion of dichloromethane (20 mL) wasadded. The reaction mixture was then transferred to a separatory funneland the organic layer was washed successively with saturated aqueoussodium bicarbonate, water, and brine. The organic layer was dried(sodium sulfate), filtered, and the filtrate solvent removed in vacuo.The crude product so obtained was purified by silica gel columnchromatography (6% EtOAc in Hexane) to yield 2.53 g (2.13 mmol, 29%) of6 as an oil. ¹ H NMR (300 MHz, CDCl₃) d 7.67-7.34 (m, 20H), 5.37 (m,4H), 5.03 (m, 1H), 4.29 (dd, J=3, 12 Hz, 1H), 4.06 (dd, J=6, 12 Hz, 1H),3.65 (t, J=6, 4H), 2.67 (m, 6H), 2.23 (m, 4H), 2.02 (m, 8H), 1.51 (m,4H), 1.29 (m, 40), 1.05 (s, 18H), 0.90 (t, J=5 Hz, 6H); ¹³ C NMR (75MHz, CDCl₃) d 173.3, 172.9, 135.5, 133.6, 130.0, 129.8, 129.7, 129.6,127.6, 127.5, 70.0, 63.5, 62.5, 57.0, 55.4, 34.3, 34.05, 31.9, 30.0,29.8, 29.7, 29.5, 29.4, 29.3, 29.2, 29.1 (2), 27.4, 27.2, 27.0, 26.8,24.9 (2), 22.7, 19.1, 14.1; IR (KBr) 3071, 2927, 1741 cm⁻¹.

Example 6 (±)-3-N,N-Bis(2-hydroxyethyl)amino!-1,2-bis(9(z)octadecenoyloxy)propane,Compound 7 in an above Specific Synthesis Scheme

To a solution of amine 6 (2.50 g, 2.10 mmol) in THF (11 mL) at 0° C. wasadded dropwise a solution of tetrabutylammonium fluoride (6 mL of a 1Msolution in THF, 6 mmol). The reaction was stirred at 0° C. for 15 h atwhich time analysis by thin layer chromatography revealed that nostarting material was present. The reaction mixture was diluted withdichloromethane and transferred to a separatory funnel. The reactionmixture was washed sequentially with saturated aqueous sodiumbicarbonate, water, and brine. The resultant organic layer was driedover sodium sulfate, filtered and the filtrate solvent removed in vacuo.The crude product was passed through a short column of silica gel using5% methanol in methylene chloride to yield 1.03 g (1.45 mmol, 69%) of 7as an oil. ¹ H NMR (300 MHz, CDCl₃) d 5.34 (m, 4H), 5.18 (m, 1H), 4.36(dd, J =3, 12 Hz, 1H), 4.10 (dd, J=6, 12 Hz, 1H), 3.60 (t, J=5 Hz, 4H),2.71 (m, 6H), 2.32 (dd, J=7, 14 Hz, 4H), 2.00 (m=8H), 1.61 (m, 4H),1.37-1.15 (m, 40H), 0.87 (t, J=6 Hz, 6H); ¹³ C NMR (75 MHz, CDCl₃) d173.7, 173.5, 129.9, 129.7, 129.6, 70.0, 63.5, 59.8, 57.2, 55.8, 34.3,34.0, 31.9, 29.7 (2), 29.6 (2), 29.5, 29.4, 29.3, 29.1 (2), 27.2, 27.1,24.8, 22.6, 14.1; IR (KBr) 3416, 2925, 1740 cm⁻¹.

Example 7 (±)-N,N- Bis(2-hydroxyethyl)!-N-methyl-N-2,3-bis(9(z)-octadecenoyloxy)propyl!ammonium chloride (DODHP), Compound1 in an above Specific Synthesis Scheme

To a sealed tube containing amine 7 (0.40 g, 0.56 mmol) was addediodomethane (3 mL). The tube was flushed with argon then sealed. Thereaction mixture was heated to 80° C. for 15 h. After this time, thereaction mixture was concentrated under a stream of argon (Caution:perform evaporation in a fume hood). The resulting yellow oil wasdissolved in methylene chloride and transferred to a round bottomedflask. This mixture was concentrated by rotary evaporation to insurethat all residual iodomethane was removed. The crude product was passedthrough a short silica gel column (gradient, 5%-10% methanol indichloromethane) to yield 0.47g (0.55 mmol, 98%) of 1 as a wax. ¹ H NMR(300 MHz, CDCl₃) d 5.69 (m, 1H), 5.32 (m, 4H), 4.47 (dd, J=3, 12 Hz,1H), 4.25-4.12 (m, 5H), 3.95-3.76 (m, 6H), 3.36 (s, 3H), 2.57 (s, 2H),2.37 (m, 4), 1.99 (m, 8H), 1.58 (m, 4H), 1.37-1.24 (m, 40H), 0.86 (t,J=6 Hz, 6H); ¹³ C NMR (75 MHz, CDCl₃) d 173.2, 172.7, 129.9, 129.5 (2),65.5 (2), 63.9, 63.3, 55.6, 51.2, 34.2, 33.9, 31.8, 29.7, 29.5, 29.4,29.2, 29.1, 29.0 (2), 27.1, 24.7, 24.6, 22.6, 14.0); IR (KBr).

Example 8 (±)-1-(Triphenylmethoxy)-3- NN-bis(2-methoxyethyl)amino!-2-propanol, Compound 10 in an above SpecificSynthesis Scheme

To a mixture of oxirane 3 (5.00 g, 15.8 mmol) and lithium perchlorate(3.36 g, 31.6 mmol) in absolute ethanol (80 mL) was added amine 9 (2.53g, 19.0 mmol). The reaction mixture was warmed to 65° C. and allowed tostir for 24 h. After this time, the reaction solution was allowed tocool to room temperature and then transferred to a separatory funnelcontaining diethylether (20 mL). The resultant mixture was sequentiallywashed with saturated aqueous sodium bicarbonate, water, and brine. Theorganic layer was dried over sodium sulfate, filtered and the filtratewas concentrated by rotary evaporation to give the crude product as ayellow oil. Purification was accomplished by SiO₂ column chromatography(3% methanol in dichloromethane) to yield 6.46 g (14.18 mmol, 90%) of 10as an oil. ¹ H NMR (300 MHz, CDCl₃) d 7.49-7.22 (m, 15H), 3.82 (m,1H),3.44 (m, 4H), 3.34 (s, 6H), 3.22 (dd, J=6, 9 Hz, 1H), 3.06 (dd, J=6, 9Hz,1H), 2.89-2.71 (m, 5H), 2.57 (dd, J=9, 13 Hz, 1H); ¹³ C NMR (75 MHz,CDCl₃) d 144.0, 128.7, 127.7, 126.9, 71.2, 67.8, 66.0, 58.7, 58.4, 54.7;IR (KBr) 3437, 3058, 2874, 1449 cm⁻¹.

Example 9 (±)-3- N,N-Bis(2-methoxyethyl)amino!-1,2-propanediol, Compound11 in an above Specific Synthesis Scheme

To a mixture of amine 10 (2.86 g, 6.28 mmol) in diethylether (13.4 mL)was added 85% formic acid (16.7 mL). The resulting reaction mixture wasstirred at room temperature for 20 h. After this time, NaHCO₃ was addedto neutralize the acidic solution. The resultant mixture wassubsequently diluted with diethylether () and transferred to aseparatory funnel. The organic layer was separated and sequentiallywashed with water, brine, and dried (sodium sulfate). Purification wasaccomplished by SiO₂ column chromatography (3% methanol indichloromethane) to yield 0.99 g (4.64 mmol, 74%) of 11 as an oil. ¹ HNMR (300 MHz, CDCl₃) d 3.68 (m, 2H), 3.53-3.39 (m, 6H), 3.33 (s, 6H),2.86-2.70 (m, 5H), 2.64 (d, J=6 Hz, 2H); ¹³ C NMR (75 MHz, CDCl₃) d71.1, 68.7, 64.7, 58.7, 57.7, 54.8; IR (KBr) 3413, 2876 cm⁻¹.

Example 10 (±)-3-N,N-Bis(2-methoxyethyl)amino!-1,2-bis(9(z)-octadecenoyloxy)propane,Compound 12 in an above Specific Synthesis Scheme

To a mixture of diol 11 (0.30 g, 1.41 mmol), triethylamine (0.5 mL), and4-dimethylaminopyridine (17.2 mg, 0.14 mmol) in dichloromethane (14 mL)at 0° C. was added dropwise oleoyl chloride (1.10 g, 3.66 mmol). Oncomplete addition, the reaction mixture was allowed to stir at 0° C. for4 h whereupon an additional portion of dichloromethane (10 mL) wasadded. The reaction mixture was then transferred to a separatory funneland the organic layer was washed successively with saturated aqueoussodium bicarbonate, water, and brine. The organic layer was dried(sodium sulfate), filtered, and the filtrate solvent removed in vacuo.The crude product so obtained was purified by silica gel columnchromatography (1% methanol in dichloromethane) to yield 150 mg (0.21mmol, 15%) of 12 as an oil. ¹ H NMR (300 MHz, CDCl₃) d 5.31 (m, 4H),5.08 (m, 1H), 4.35 (dd, J =3, 12 Hz, 1H), 4.09 (dd, J=6, 12 Hz, 1H),3.40 (t, J=6 Hz, 4H), 3.29 (s, 6H), 2.76-2.68 (m, 6H), 2.26 (m, 4H),1.98 (m, 8H), 1.58 (m, 4H), 1.35-1.22 (m, 40H), 0.85 (t, J=6 Hz, 6H); ¹³C NMR (75 MHz, CDCl₃) d 173.3, 173.0, 129.9, 129.6, 71.4, 70.1, 63.7,58.7, 55.2. 54.8, 34.3, 34.1, 31.8, 29.7, 29.6, 29.5, 29.2, 29.1, 29.0,27.0 (2), 24.8, 22.6,14.0; IR (KBr) 2925, 2854, 1740 cm⁻¹.

Example 11 (±)-N,N-Bis(2-methoxyethyl)-N-methyl-N-2,3-bis(9(z)-octadecenoyloxy)propyl!ammonium chloride (DODMP), Compound8 in an above Specific Synthesis Scheme

To a sealed tube containing amine 12 (150 mg, 0.20 mmol) was addediodomethane (3 mL). The tube was flushed with argon then sealed. Thereaction mixture was heated to 80° C. for 15 h. After this time, thereaction mixture was concentrated under a stream of argon (Caution:perform evaporation in a fume hood). The resulting yellow oil wasdissolved in methylene chloride and transferred to a round bottomedflask. This mixture was concentrated by rotary evaporation to insurethat all residual iodomethane was removed. The crude product was passedthrough a short silica gel column (gradient, 5%-10% methanol indichloromethane) to yield 162 mg (0.19 mmol, 95%) of 8 as a wax. ¹ H NMR(300 MHz, CDCl₃) d 5.59 (m, 1H), 5.24 (m, 4H), 4.40 (dd, J=3, 12 Hz,1H), 4.13-3.75 (m, 11H), 3.34 (m, 9H), 2.25 (m, 4H), 1.91 (m, 8H), 1.51(m, 4H), 1.27-1.15 (m, 40H), 0.78 (m, 6H); ¹³ C NMR (75 MHz, CDCl₃) d172.8, 172.6, 129.8, 129.4, 129.4, 65.9 (2), 63.5, 63.2, 63.0, 59.2,50.4, 34.1, 33.8, 31.7, 29.5 (2), 29.3, 29.2 (2), 29.1, 29.0, 28.9 (2),29.8, 27.0 (2), 24.5, 24.4, 22.5, 13.9; IR (KBr) 3004, 2925, 1744 cm⁻¹.

Example 12 (±)-N-(2,2,2-Trifluoroethyl)-N,N-dimethyl-N-2,3-bis(9(z)-octadecenyloxy)propyl!ammonium chloride (DOFEP), Compound14 in an above Specific Synthesis Scheme

To a sealed tube containing amine 15 (0.50 g, 0.77 mmol) in DMF (5 mL)was added 2-iodo-1,1,1-trifluoroethane (1.1 mL). The tube was flushedwith argon then sealed. The reaction mixture was heated to 100° C. for15 h. After this time, the reaction mixture was transferred round bottomflask and the volatiles (DMF, excess ICH₂ CF₃) were removed viadistillation at reduced pressure. The resulting yellow oil was passedthrough a short silica gel column (gradient, 5%-10% methanol indichloromethane) to yield 67 mg (0.07 mmol, 10%) of 14 as a solid. ¹ HNMR (300 MHz, CDCl₃) d 5.59 (m, 1H), 5.33 (m, 4H), 4.51 (m, 2H), 4.13(dd, J=6, 12, 1H), 3.87 (dd, J=9, 14 Hz, 1H), 3.53 (s, 6H), 2.35 (m,4H), 1.99, (m, 8H), 1.59 (m, 4H), 1.29-1.25 (m, 40H), 0.87 (t, J=7 Hz,6H);; ¹³ C NMR (75 MHz, CDCl₃) d 173.0, 172.5, 129.9, 129.8, 129.5,129.4, 66.0, 65.6, 62.8, 54.6, 34.1, 33.8, 31.7, 39.7, 29.6, 29.4 (2),29.3, 29.1 (2), 29.0 (2), 28.9, 27.1, 27.0, 24.6, 2.45, 22.5, 13.9); IR(KBr).

Example 13 Liposome Formulation

An appropriate mass of the cationic lipid and a neutral lipid (DOPE)were added as solutions in chloroform to 1.9 mL sample vials to yield a50:50 molar ratio of cationic lipid:neutral lipid. The chloroform wasremoved via rotary evaporation at 37° C. The resulting thin lipid filmswere placed under vacuum overnight to insure that all traces of solventhave been removed. The lipid mixture was resuspended in 1 mL sterilewater at 70° C. until the film is hydrated, and then vortex mixed toafford an emulsion (unsonicated preparation). These emulsions wereformulated at a cationic lipid concentration of 1 mM. To form thesonicated preparations used in this study, the lipid emulsions weresonicated using a Branson sonifier 450 sonicator equipped with a cuphorn and recirculating water bath (35° C., 80% output with 2 sec delaysover 15 minutes.). By performing comparative transfection experiments,it was determined that sonication of cytofectin emulsions above theirphase transition temperature did not significantly alter transfectionefficacy. Furthermore, sonication at or above 70° C. resulted in partiallipid decomposition as determined by thin layer chromatography.

Example 14 Cell Culture

NIH 3T3 cells were obtained from ATCC (CRL 1658), cultured in Dulbecco'sModified Eagle's Medium with 10% calf serum, and plated on standard 24well tissue culture plates 12 to 24 hours prior to transfection. Cellswere approximately 80% confluent at the time of transfection. CHO cells(ATCC CCL 61) were cultured using Ham's F12 medium supplemented with 10%fetal calf serum, and plated as described for NIH 3T3.

Example 15 Transfection of Cultured Cells

NIH 3T3 cells were plated onto 24 well tissue culture plates asdescribed above. The growth media was removed via aspiration and thecells were washed once with 0.5 mL PBS/well. The liposome/DNA complexeswere formed through sequential addition of appropriate amounts of DMEM(serum-free), plasmid DNA (4 micrograms), and the liposome formulationinto a 2 mL Eppendorf tube to a total volume of 800 microliters.Typically, 24 microliters of a lipid emulsion (1 mM cytofectin, 1 mMDOPE) were used to complex 4 micrograms of DNA to yield a 2:1 cytofectinto DNA molar charge ratio. The addition of these substances was followedby thorough vortex mixing and incubation for 15 minutes at roomtemperature. A 200 microliter aliquot of the resultant transfectioncomplex was added to each well (1 microgram DNA/well, n=4) and the cellswere incubated for 4 hrs. at 37° C. At this time, 500 microliters of theappropriate growth media +10% calf serum/well was added and the cellscultured for approximately 48 hours prior to lysis and analysis. Thesample transfections were subsequently repeated a minimum of three timesfor each cell line in order to ensure reproducibility.

Example 16 Intratracheal Instillation of DNA or Lipid/DNA Complexes

Female Balb/C mice (specific pathogen free) weighing approximately 20 to21 grams were obtained from Charles River Laboratories. Anesthesia wasprovided for invasive procedures and animals were terminated by CO₂inhalation in accordance with University of California, Davisguidelines. DNA was prepared for instillation by dilution in sterilewater. Lipid/DNA complexes were prepared by mixing 20 micrograms ofplasmid DNA (Luciferase) at a 4:1 molar charge ratio (cationiclipid:DNA) in sterile water for injection (total volume of 240microliters). Mixtures were prepared and vortex mixed at roomtemperature, and injected within 5 minutes of lipid:DNA complexformation. Neck dissections were performed on anesthetized mice using a1 cm incision through the skin of the anterior neck, dissection of thesalivary gland and musculature surrounding the anterior tracheaimmediately below the larynx, and instillation of 240 microliters of DNAor lipid/DNA complex using a 1/2" 30 g needle inserted 1-3 tracheal ringinterspaces inferior to the larynx. After injection, the salivary glandwas placed over the tracheal defect, and the superficial neck woundclosed with staples. Mice were killed 48 hours after treatment and atracheal/lung block dissected, homogenized in lysis buffer, and assayedfor luciferase protein as described below. Mock treated mouselung/trachea was used for assessment of background luciferase activity.No activity was detected in control mock-treated mouse tissue.

Example 17 Luciferase Assay

Relative luciferase activity was determined by using the EnhancedLuciferase Assay Kit and a Monolight 2010 luminometer (both fromAnalytical Luminescence Laboratories, San Diego, Calif.). This wasaccomplished by directly applying 233.3 mL of concentrated luciferaselysis buffer (final concentration 0.1M potassium phosphate pH 7.8, 1%Triton X100, 1 mM DTT, 2 mM EDTA) to each well and placing the cells onice for 15 minutes. Removal of growth media was not necessary prior tothe application of the lysis buffer. This technique enhancesreproducibility by avoiding the possibility of cell loss during mediaremoval. An analogous experiment where the growth media was removedafforded similar results. Luciferase light emissions from 31 mL of thelysate were measured over a 10 second period, and results were expressedas a function of an assumed total lysate volume of 933.3 mL. Activityhas been expressed as relative light units, which are a function ofassay conditions, luciferase concentration, luminometer photomultipliertube sensitivity and background. Under the conditions described above,relative light units are related to luciferase protein mass by theequation fg luciferase=(RLU/48.6) -824!.

Example 18 Generation of Counterion Species

A panel of DOTAP analogs was prepared by altering the anionic counterionthat accompanies the ammonium head group using ion-exchangechromatography. N-(1-(2,3-Dioleoyloxy)propyl)-N,N,N-trimethylammoniumiodide (DOTAP) was prepared in a similar manner to the Silivius method(Leventis, R. and Silvius, J. R. (1990) Fiochim. Biophys. Acta 1023,124-132). Chloride substitution was achieved using Dowex stronglyanionic exchange resin, 8% crosslink (200-400 mesh), chloride form.Acetate substitution was performed using the anionic exchange resinAG1-8X (200-4-mesh), acetate form, and the remaining counterions wereobtained using the hydroxide form of this resin. The chloride andacetate ion exchange was performed by suspending the corresponding resin(1.0-1.5 g) in highly purified filtered water (10-20 ml), and loadinginto a narrow bore glass column. The column was washed with water (5×, 5ml), methanol (10×, 5 ml), and equilibrated with a CH₃ OH--CH₂ Cl₂ (8:2)solution. A solution of the cationic lipid (50-70 mg lipid in ca. 1 mlCH₃ 0H--CH₂ Cl₂ (8:2)) was then gravity eluted through the column. Forthe substitution of all other counterions, the hydroxide resin waspretreated by washing with a 1M solution of the desired counterion asits sodium salt. The loaded resin was then washed with water until theeluent pH stabilized at approximately 7. The ion exchange chromatographywas then performed using the CH₃ OH--CH₂ Cl₂ equilibration sequencedescribed above. Electrospray ionization mass spectrometry was used toverify the composition of the resulting salt forms.

The influence of these counterions on transfection was studied by usinglipid films and lipid-DNA complexes that were prepared, transfected, andsubsequently analyzed as previously described (Ruysschaert, J. M., elOuahabi, A., Willeaume, V., Huez, G., Fuks, R., Vandenbranden, M. and DiStefano, P. 1994. A novel cationic amphiphile for transfection ofmammalian cells. Biochem. Biophys. Res. Commun. 203(3): 1622-8). Theplasmid pNDCLux, which encodes the P. pyralis luciferase, was

Example 19 Transfection Data

Interpretation of the various figures is facilitated by reference to theabove described compounds and to the following list of abbreviations,prefixes, or suffixes and the associated structures:

    ______________________________________    2 #STR21##    --TAP    3 #STR22##    --EAP    4 #STR23##    --PAP    5 #STR24##    --RI    6 #STR25##    --MEP    7 #STR26##    --FEP    8 #STR27##    --DEP    9 #STR28##    --TEP    0 #STR29##    --DHP    1 #STR30##    --DMP    ______________________________________    DO     as a prefix for the above "R" indicates dioleoyl (with the           carbonyl)    DM     as a prefix for the above "R" indicated dimyristoyl (with           the carbonyl)    DOPE   Dioleoylphosphatidylethanolamine    DOTMA  N- 1-(2,3-dioleyloxy)propyl!-N,N,N-trimethylammonium           bromide  DIETHER!    ______________________________________

FIG. 1 shows a comparison of cytofectin-mediated DNA transfection usingNIH 3T3 cells. DNA transfections were performed in quadruplicate asdescribed in the experimental procedures using a 2:1 molar charge ratio(lipid charge to DNA phosphate charge). The data demonstrates that theincorporation of a dihydroxyethyl substituted ammonium functionality inthe lipid polar domain leads to significantly higher transfectionefficacy in vitro. Results are summarized in bar graph form as the mean(n=4) and standard deviation of total luciferase light units (RLU)obtained from cells lysed after transfection of 1 microgram of DNA. Allcytofectins were formulated at a 1:1 molar ratio with DOPE.

In FIG. 2 there is shown an in vivo comparison of cytofectin-mediatedDNA transfection. Balb-C mice were transfected with plasmid DNA usingvarious cytofectins. Intratracheal instillations of cytofectin:DNAcomplexes were performed as described in the experimental procedures.The data demonstrates that the incorporation of dimethoxyethyl andtriflouroethyl substituted ammonium functionality in the lipid polardomain leads to significantly higher transfection efficacy in vivo.Results are summarized in bar graph form as the mean (n=4) and standarddeviation of total luciferase light units (RLU) obtained fromtrachea/lung blocks lysed 48 hours after treatment with 20 micrograms ofDNA.

The transfection activity of various compounds is illustrated in FIG. 3.As can be seen in FIG. 3, the dioleoyl derivatives of -FEP and DMP ofthe subject invention are exceptionally effective in transfection.General transfection conditions were as above.

For the comparison of cytofectin polar domain structure to transfectionactivity in NIH 3T3 cells shown in FIG. 6, liposome formulationscontaining a 1:1 mole ratio of Cytofectin and DOPE were mixed with 1 mgof pNDCLUX plasmid DNA to give a 2:1 molar charge ratio (lipid charge toDNA phosphate). The resultant complex was placed directly on to the cellsurface. Cell lysates obtained 48 hours after transfection were analyzedfor luciferase activity. Each data point reflects the mean value oftotal light units derived from four transfections and the standarddeviation from this mean.

FIG. 7 depicts intratracheal instillation into mice. For theintratracheal instillation into mice the dioleoyl hydrophobic domainsare more effective than corresponding dimyristoyl analogs. Also,cytofectins incorporating inductive functionality enhance mouseintratracheal transfection.

For the comparison of cytofectin counterions to transfection activity inNIH 3T3 cells depicted in FIG. 8, liposome formulations containing a 1:1mole ratio of Cytofectin and DOPE were mixed with 1 mg of pNDCLUXplasmid DNA to give a 2:1 molar charge ratio (lipid charge to DNAphosphate). The resultant complex was placed directly on to the cellsurface. Cell lysates obtained 48 hours after transfection were analyzedfor luciferase activity. Each data point reflects the mean value oftotal light units derived from four transfections and the standarddeviation from this mean.

For the comparison of cytofectin counterions to in vivo transfectionactivity in Balb-C lung illustrated in FIG. 9, Balb-C mice weretransfected with lipid/DNA complexes formed from mixing pNDCLUX plasmidDNA with various cytofectins (2:1 lipid to DNA phosphate charge ratio).Intratracheal installations of cytofectin:DNA complexes were performedas described elsewhere (Balasubramaniam, R. P., Bennett, M. J., Aberle,A. M., Malone, J. G., Nantz, M. H. and Malone, R. W. 1996. Structuraland functional analysis of cationic transfection lipids: the hydrophobicdomain. Gene Ther. 3(2): 163-172). Results are summarized in bar graphform as the mean (n=4) and standard deviation of total luciferase lightunits obtained from trachea/lung blocks lysed 48 hours after treatmentwith 20 mg of DNA.

For the comparison of cytofectin hydrophobic structure to transfectionactivity in NIH 3T3 cells shown in FIG. 10, liposome formulationscontaining a 1:1 mole ratio of Cytofectin and DOPE were mixed with 1 mgof pCMVL plasmid DNA to give a 2:1 molar charge ratio (lipid charge toDNA phosphate). The resultant complex was placed directly on to the cellsurface. Cell lysates obtained 48 hours after transfection were analyzedfor luciferase activity. Each data point reflects the mean value oftotal light units derived from four transfections and the standarddeviation from this mean.

For the comparison of cytofectin hydrophobic structure to transfectionactivity in human bronchial epithelial cells (16HBE14o-) presented inFIG. 11, liposome formulations containing a 1:1 mole ratio of Cytofectinand DOPE were mixed with 1 mg of pCMVL plasmid DNA to give a 2:1 molarcharge ratio (lipid charge to DNA phosphate). The resultant complex wasplaced directly on to the cell surface. Cell lysates obtained 48 hoursafter transfection were analyzed for luciferase activity. Each datapoint reflects the mean value of total light units derived from fourtransfections and the standard deviation from this mean.

For the effects of formulation conditions on luciferase expression inmurine lung disclosed in FIG. 12, an overall increase in luciferaseexpression was noted for sonicated (DOTAP Bisulfate)-DNA complexes withincreasing DNA concentrations at a fixed 2:1 charge ratio. Sonicatedcomplexes were prepared as described previously (see Table 3, above).Naked DNA has been provided as a control comparison. Included areexamples illustrating the effect of sonication with heating on both theactive lipid DODMP.Chloride as well as DOTAP.Bisulfate, a widely usedcationic transfection lipid.

The invention has now been explained with reference to specificembodiments. Other embodiments will be suggested to those of ordinaryskill in the appropriate art upon review of the present specification.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A cytofectin:polynucleotide complex comprising:a)a polynucleotide and b) a cytofectin having the structure: ##STR31##wherein m=1-10; R₁ is a hydrogen, an alkyl group, an alkenyl group, analkynyl group, a hydroxylated alkyl, alkenyl, or alkynyl group, an ethercontaining alkyl, alkenyl, or alkynyl group, or a trihalogenated alkyl,alkenyl, or alkynyl group; R₂ is an alkyl group, an alkenyl group, analkynyl group, a hydroxylated alkyl, alkenyl, or alkynyl group, an ethercontaining alkyl, alkenyl, or alkynyl group, or a trihalogenated alkyl,alkenyl, or alkynyl group R₃ is an alkyl group, an alkenyl group, analkynyl group, a hydroxylated alkyl, alkenyl, or alkynyl group, an ethercontaining alkyl, alkenyl, or alkynyl group, or a trihalogenated alkyl,alkenyl, or alkynyl group, however, when two of R₁, R₂ and R₃ are alkylsor alkenyls then the other is a trihalogenated group or when R₁ ishydrogen and R₂ is an alkyl or alkenyl then R₃ is a trihalogenatedgroup; R₄ is an alkyl group, an alkenyl group, an alkynyl group, or analkyl, alkenyl, or alkynyl containing acyl group; R₅ is an alkyl group,an alkenyl group, an alkynyl group, or an alkyl, alkenyl, or alkynylcontaining acyl group; and X⁻ is a counterion.
 2. A compositionaccording to claim 1, wherein said counterion is selected from a groupconsisting of the oxyanions bisulfate or trifluoromethanesulfonate andthe halides iodide or bromide.
 3. A cytofectin:polynucleotide complexproduced by the steps comprising:a) mixing a cytofectin with apolynucleotide, wherein said cytofectin comprises a composition ofmatter having the structure: ##STR32## wherein m=1-10; R₁ is a hydrogen,an alkyl group, an alkenyl group, an alkynyl group, a hydroxylatedalkyl, alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, or a trihalogenated alkyl, alkenyl, or alkynyl group; R₂is an alkyl group, an alkenyl group, an alkynyl group, a hydroxylatedalkyl, alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, or a trihalogenated alkyl, alkenyl, or alkynyl group R₃is an alkyl group, an alkenyl group, an alkynyl group, a hydroxylatedalkyl, alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, or a trihalogenated alkyl, alkenyl, or alkynyl group,however, when two of R₁, R₂, and R₃ are alkyls or alkenyls then theother is a trihalogenated group or when R₁ is hydrogen and R₂ is analkyl or alkenyl then R₃ is a trihalogenated group; R₄ is an alkylgroup, an alkenyl group, an alkynyl group, or an alkyl, alkenyl, oralkynyl containing acyl group; R₅ is an alkyl group, an alkenyl group,an alkynyl group, or an alkyl, alkenyl, or alkynyl containing acylgroup; and X⁻ is an anion and b) energizing said mixture of cytofectinand polynucleotide for a selected period of time at a predeterminedtemperature to produce a thermodynamically stable product.
 4. Acytofectin:polynucleotide complex produced by the steps comprising:a)combining a cytofectin with a lipid, wherein said cytofectin comprises acomposition of matter having the structure: ##STR33## wherein m=1-10; R₁is a hydrogen, an alkyl group, an alkenyl group, an alkynyl group, ahydroxylated alkyl, alkenyl, or alkynyl group, an ether containingalkyl, alkenyl, or alkynyl group, or a trihalogenated alkyl, alkenyl, oralkynyl group; R₂ is an alkyl group, an alkenyl group, an alkynyl group,a hydroxylated alkyl, alkenyl, or alkynyl group, an ether containingalkyl, alkenyl, or alkynyl group, or a trihalogenated alkyl, alkenyl, oralkynyl group R₃ is an alkyl group, an alkenyl group, an alkynyl group,a hydroxylated alkyl, alkenyl, or alkynyl group, an ether containingalkyl, alkenyl, or alkynyl group, or a trihalogenated alkyl, alkenyl, oralkynyl group, however, when two of R₁, R₂, and R₃ are alkyls oralkenyls then the other is a trihalogenated group or when R₁ is hydrogenand R₂ is an alkyl or alkenyl then R₃ is a trihalogenated group; R₄ isan alkyl group, an alkenyl group, an alkynyl group, or an alkyl,alkenyl, or alkynyl containing acyl group; R₅ is an alkyl group, analkenyl group, an alkynyl group, or an alkyl, alkenyl, or alkynylcontaining acyl group; and X⁻ is an anion; b) mixing said combination ofcytofectin and lipid with polynucleotide; and c) sonicating said mixtureof polynucleotide, cytofectin, and lipid for a selected period of timeat a predetermined temperature.
 5. A method for producing a transfectionactive cytofectin:polynucleotide complex comprising the steps:a)combining a cytofectin having a quaternized amine and a counterionselected from a group consisting of the oxyanions bisulfate ortirfluoromethanesulfonate and the halides iodide or bromide with alipid, wherein said cytofectin comprises a composition of matter havingthe structure: ##STR34## wherein m=1-10; R₁ is a hydrogen, an alkylgroup, an alkenyl group, an alkynyl group, a hydroxylated alkyl,alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, or a trihalogenated alkyl, alkenyl, or alkynyl group; R₂is an alkyl group, an alkenyl group, an alkynyl group, a hydroxylatedalkyl, alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, or a trihalogenated alkyl, alkenyl, or alkynyl group R₃is an alkyl group, an alkenyl group, an alkynyl group, a hydroxylatedalkyl, alkenyl, or alkynyl group, an ether containing alkyl, alkenyl, oralkynyl group, or a trihalogenated alkyl, alkenyl, or alkynyl group,however, when two of R₁, R₂, and R₃ are alkyls or alkenyls then theother is a trihalogenated group or when R₁ is hydrogen and R₂ is analkyl or alkenyl then R₃ is a trihalogenated group; R₄ is an alkylgroup, an alkenyl group, an alkynyl group, or an alkyl, alkenyl, oralkynyl containing acyl group; R₅ is an alkyl group, an alkenyl group,an alkynyl group, or an alkyl, alkenyl, or alkynyl containing acylgroup; and X⁻ is said counterion and is selected from a group consistingof the oxyanions bisulfate or trifluoromethanesulfonate and the halidesiodide or bromide; b) mixing said combination of cytofectin and lipidwith polynucleotide; and c) sonicating said mixture of polynucleotide,cytofectin, and lipid for a selected period of time at a predeterminedtemperature.